EP0753152B1 - Festphasenimmunoassay zum nachweis von inhibitoren von einem proteolytischen enzym - Google Patents

Festphasenimmunoassay zum nachweis von inhibitoren von einem proteolytischen enzym Download PDF

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Publication number
EP0753152B1
EP0753152B1 EP95913069A EP95913069A EP0753152B1 EP 0753152 B1 EP0753152 B1 EP 0753152B1 EP 95913069 A EP95913069 A EP 95913069A EP 95913069 A EP95913069 A EP 95913069A EP 0753152 B1 EP0753152 B1 EP 0753152B1
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Prior art keywords
tubulin
antibody
support
peptide
linked
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French (fr)
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EP0753152A1 (de
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Khalid Islam
Lucia Carrano
Maurizio Denaro
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Gruppo Lepetit SpA
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Gruppo Lepetit SpA
Lepetit SpA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/815—Test for named compound or class of compounds
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/811—Peptides or proteins is immobilized on, or in, an inorganic carrier
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/81—Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
    • Y10S530/812—Peptides or proteins is immobilized on, or in, an organic carrier
    • Y10S530/815—Carrier is a synthetic polymer
    • Y10S530/816—Attached to the carrier via a bridging agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/839—Nerves; brain

Definitions

  • the present invention refers to a solid phase immunoassay for detecting specific inhibitors of proteolytic enzymes in biological fluids or in any kind of solution containing them, as well as for detecting proteolytic activities in any solution containing them.
  • the assay of the invention comprises contacting a tubulin protein or a tubulin-like peptide covalently linked to a suitable support with a solution containing a protease together with a related inhibitor and determining said inhibitor by using a monoclonal antibody which specifically recognizes the free end of the linked tubulin.
  • tubulin-like peptide is comprised any peptide on which the more common classes of proteases are active and the free end of which is identical to the free end of tubulin.
  • free end it is intended the end of the tubulin protein or of the peptide which is not linked to the support; as said protein or peptide is linked to the support preferably via its N-terminus, in general the “free end” corresponds to the C-terminus end of the amino acid sequence.
  • the method of the present invention may also be employed to detect the presence of the above proteases in the tested solution, by detecting them with specific known inhibitors.
  • the method of the invention is accurate, precise, rapid and easy to practice.
  • the intra- and inter- assay precision are well within the range of values currently accepted for analytical purposes.
  • the activity of the inhibitors can also be determined when any impurity is present in the tested solution, as after the enzymatic reaction the peptidic substrate is thoroughly washed before detecting the activity of the inhibitor.
  • kits for use in the above method for determining anti-proteolytic activities which comprises a support to which a tubulin protein or a tubulin-like peptide is covalently linked and a solution containing a monoclonal antibody which specifically recognizes the free end of the linked tubulin.
  • Preferred proteases for which the method of the present invention is particularly useful are the proteases of the serine, aspartic and cysteine classes.
  • the peptidic substrate which is employed in the assay of the present invention for determining the inhibition of anti-proteolytic substances against the above proteases is tubulin or a tubulin-like peptide.
  • animal tubulin is employed, particularly preferred being bovine brain tubulin.
  • bovine brain tubulin may be purified from bovine brain extract according to Islam K. and R.G Burns, FEBS Lett., 123, 1981, pp. 181-185 and R.G Burns and Islam K., Europ. Jour. Biochem., 117, 1981, 515-519; separation of tubulin from microtubule-associated proteins (MAPs) may be performed by known chromatographic procedures, preferably by ion-exchange chromatography according to Vallee et al. in "Methods in Enzymology", 134, 1986, pp. 89-116.
  • MAPs microtubule-associated proteins
  • tubulin is obtained, and employed in the present assay, as an equimolar mixture of ⁇ - and ⁇ -tubulin. Both these proteins have suitable digestion sites for the more common classes of proteases, but they differ for the amino acid sequence of the C-terminus end of the protein.
  • the specific monoclonal antibody used for detecting the undigested protein preferably a monoclonal antibody which specifically recognizes the C-terminus of ⁇ -tubulin, will recognize only one of the two undigested tubulin proteins; this difference is however of no practical relevance for the results of the assay of the present invention, because of the constant equimolarity of the ⁇ - and ⁇ -tubulin mixture employed.
  • the predicted amino acid sequences of ⁇ -tubulin molecules are disclosed in Molec Cell Biol 6 (1986) 906-913.
  • tubulin As it readily appears to the skilled man not only tubulin but also any degradation product of tubulin or any modified tubulin may be employed for the immunoassay of the present invention, insofar as said peptide has suitable digestion sites for the more common classes of proteases, while maintaining the characteristic C-terminus end of tubulin, preferably ⁇ -tubulin.
  • any peptide on which the more common classes of proteases are active and the C-terminus end of which is identical to the C-terminus end of tubulin, preferably ⁇ -tubulin, may also be employed.
  • Said peptide shall therefore contain the characteristic digestion sites for the detected proteases.
  • peptides containing Leu, Phe or Tyr may be digested by Elastase or Chymotrypsin (Serine proteases); peptides containing Phe or Tyr may be digested by Pepsin and Cathepsin D (Aspartic proteases); peptides containing Arg or Lys may be digested by Trypsin (Serine protease); peptides containing the sequence Tyr-Pro-Leu may be digested by Renin (Aspartic protease); peptides containing Phe may be digested by Cathepsin B (Cysteine protease); peptides containing Arg, His or Gly may be digested by Papain (Cysteine protease).
  • the specific amino acid sequence of the C-terminus end of the peptide will depend upon the monoclonal antibody employed for detecting the undigested peptide. For instance, when YL 1/2 antibody is employed, the amino acid sequence of the C-terminus end of the peptide will be: -Glu-Gly-Glu-Gly-Glu-Glu-Glu-Gly-Glu-Glu-Tyr. This sequence corresponds to the sequence of 11 amino acids of the C-terminus end of ⁇ -tubulin.
  • tubulin-like peptides synthetic peptides may be employed.
  • a new synthetic peptide consisting of 21 amino acids is employed, wherein the last 11 amino acids of the C-terminus end of said peptide are identical to those of the C-terminus end of tubulin.
  • amino acid sequence of said novel peptide is:
  • Natural amino acids with the exception of glycine, contain a chiral atom of carbon.
  • amino acids of the above peptide are meant to be in their L-configuration.
  • the peptide of this invention can be prepared by a variety of procedures readily known to those skilled in the art. Such procedures include the solid phase sequential procedure, according to the method of Merrifield, which can be performed using established automated methods such as by use of an automated peptide synthesizer.
  • the resin support employed can be any suitable resin conventionally employed in the art for the solid phase preparation of polypeptides, preferably polystyrene which has been cross-linked with from 0.5 to about 3 percent divinyl benzene, which has been either converted to the p-methylbenzhydrylamine or benzhydrylamine derivative (for C-terminal amides) or chloromethylated or hydroxymethylated to provide sites for ester formation with the initially introduced ⁇ -amino protected amino acid (for producing C-terminal alkylamides) and esters.
  • suitable resin conventionally employed in the art for the solid phase preparation of polypeptides, preferably polystyrene which has been cross-linked with from 0.5 to about 3 percent divinyl benzene, which has been either converted to the p-methylbenzhydrylamine or benzhydrylamine derivative (for C-terminal amides) or chloromethylated or hydroxymethylated to provide sites for ester formation with the initially introduced ⁇ -amino protected amino acid (for
  • a 2-bromobenzyloxycarbonyl (2-BrZ) protected Tyr bound to a benzylated, hydroxymethylated phenylacetamidomethyl resin can be used and is commercially available (e.g. ABI APPLIED BIOSYSTEMS®).
  • the protecting group is removed using any suitable procedure such as by using trifluoroacetic acid in methylene chloride, trifluoroacetic acid alone, or HCl in dioxane.
  • the deprotection is carried out at a temperature of from 0°C to room temperature.
  • Other standard cleaving reagents and conditions for removal of specific ⁇ -amino protecting groups may be used.
  • the other amino protected amino acids are coupled step-wise in the desired order.
  • multiple amino acid groups may be coupled by the solution method prior to coupling with the resin supported amino acid sequence.
  • the ⁇ -amino protecting group employed with each amino acid introduced into the polypeptide sequence may be any such protecting group known in the art.
  • acyl type protecting groups such as: formyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzenesulfonyl, nitro-phenylsulfenyl, tritylsulfenyl, o-nitrophenoxyacetyl and ⁇ -chlorobutyryl;
  • aromatic urethan type protecting groups such as benzyloxycarbonyl and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzoxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, ⁇ , ⁇ -dimethyl-3,5-dimethoxybenzyloxycarbonyl and benzhydryloxycarbon
  • an appropriate coupling reagent is within the skill of the art.
  • Illustrative examples are (1) carbodiimides (e.g., N,N'-dicyclohexylcarbodiimide (DCC) and N-ethyl-N'-( ⁇ -dimethylaminopropylcarbodiimide); (2) cyanamides (e.g., N,N-dibenzylcyanamide); (3) ketenimines; (4) isoxazolium salts (e.g., N-ethyl5-phenylisoxazolium-3'-sulfonate); (5) monocyclic nitrogen containing heterocyclic amides of aromatic character containing one through four nitrogens in the ring such as imidazolides, pyrazolides, and 1,2,4-triazolides; specific heterocyclic amides that are useful include N-N-carbonyldiimidazole and N,N-carbonyl-di-1,2,4-triazole;
  • Each protected amino acid or amino acid sequence is introduced into the solid phase reactor in about a fourfold excess and the coupling is carried out in a medium of dimethylformamide: methylene chloride (1:1) or in dimethylformamide alone or preferably methylene chloride alone.
  • the coupling procedure is repeated before removal of the ⁇ -amino protecting group, prior to the coupling of the next amino acid in the solid phase reactor.
  • the success of the coupling reaction at each stage of the synthesis is monitored by the ninhydrin reaction as described by E. Kaiser et al., Analyt. Biochem. 34 , 595 (1970).
  • the peptide is removed from the resin.
  • the whole protected peptide is released either from the chloromethylated resin by ammoniolysis to obtain the protected amide, or by hydrofluoric acid to obtain the acid, or from the methylbenzhydrylamine or benzhydrylamine resins by treatment with a solution of dimethyl sulfide, p-cresol or thiocresol in liquid hydrofluoric acid, at a temperature of about 0°C.
  • suitable side chain protecting groups for tyrosine are 2-halo-benzyloxycarbonyl derivatives, such as 2-bromobenzyloxycarbonyl (2-BrZ), the carboxylic group of glutamic acid can be protected with a benzyl (Bzl) or cyclohexyl ester (Chx) group, histidine is conveniently protected with a benzyloxymethyl (Bom) or a tosyl group (Tos), while the tosyl group (Tos) is also suitable for protecting the side chain of arginine.
  • 2-halo-benzyloxycarbonyl derivatives such as 2-bromobenzyloxycarbonyl (2-BrZ
  • the carboxylic group of glutamic acid can be protected with a benzyl (Bzl) or cyclohexyl ester (Chx) group
  • histidine is conveniently protected with a benzyloxymethyl (Bom) or a tosyl group (Tos)
  • protecting group removal is done after the peptide chain synthesis is complete, as the conditions for removing the peptide from the resin are in general also suitable for removing the protecting groups of the side chains. However, the protecting groups can be removed at any other appropriate time.
  • the peptide After the peptide has been removed from the resin and deprotected, it is purified according to known chromatographic techniques, preferably by HPLC, thus obtaining the desired pure peptide.
  • the method for carrying out the immunoassay of the present invention comprises:
  • the support to which the tubulin protein or the tubulin-like peptide is linked is any analytical support to which the protein or peptide may covalently be linked, preferably via its N-terminus end.
  • analytical supports which may be employed are glass or plastic microtiter plates or test tubes, plastic sheets, nitrocellulose and nylon membranes.
  • the most preferred analytical support is represented by the wells of microtiter plates made of polymeric materials, such as polyethylene, polystyrene, or polyvinylchloride.
  • Such polymers of the microtiter plate wells may either contain functional groups able to covalently link tubulin or tubulin-like peptide, or preferably they may be activated by linking to the support a so-called "spacer arm" containing a functional group which activates the support's surface, thus allowing the linkage of the protein, preferably via the N-terminus end.
  • activating agents are carbodiimide derivatives, N-hydroxysuccinimide derivatives, e.g. N-cyclohexyl-N'-2,4'-methylmorpholinimide, sulfosuccinimide derivatives, e.g. bis(sulfosuccinimidyl)suberate.
  • bis(sulfosuccinimidyl)suberate is employed as activating agent.
  • a buffered solution containing tubulin or a tubulin-like peptide preferably from 0.001 mg/ml to 0.010 mg/ml, is contacted with said support, preferably for from 1 to 2 hours at a temperature of from 18°C to 30°C.
  • the possibly unreacted activated groups on the support are saturated by adding an excess of a non-interfering aminoacid solution, such as a buffered solution from 3% to 8% of glycine for 1 to 3 hours.
  • solution containing a proteolytic activity is intended to encompass any kind of solution containing a proteolytic activity such as fermentation broths of microorganisms, cells and microorganisms culture media, human biological fluids like plasma, urines, exudates, etc., as well as any analytical solution containing said proteases.
  • Preferred proteases of which inhibitors may be determined according to the assay of the present invention are: serine proteases such as trypsin, subtilisin, elastase and cathepsin G; aspartic proteases such as pepsin, cathepsin D, renin and Hiv-1 protease; cysteine proteases such as papain and cathepsin B.
  • the protease inhibitor tested on the selected proteolytic solution may be either a compound with a known inhibitory activity (in this case a calibration curve is obtained, concentration vs. percentage of inhibition) or an unknown substance.
  • a known inhibitory activity in this case a calibration curve is obtained, concentration vs. percentage of inhibition
  • an unknown substance in the latter case, it is possible to determine the inhibition activity of the investigated compound by reference with the activity of known inhibitors; furthermore, if the unknown inhibitor has been obtained in admixture with impurities, it is also possible to follow the purification of the active substance by repeating the assay at the different steps of the purification, thus observing an increase in the inhibition activity depending on the higher degree of purity of the compound.
  • labelled monoclonal antibody it is intended a labelled antibody which specifically recognises the free-end of the linked tubulin as well as a system which comprises an antibody and a labelled anti-antibody, wherein the first antibody specifically recognises the free-end of the linked tubulin, while the labelled anti-antibody is specific for the first antibody; in general, a species specific labelled anti-antibody is conveniently employed. In both cases, antibodies which specifically recognise the C-terminus end of ⁇ -tubulin are preferably employed.
  • the labelling of the antibody is made according to known per se techniques, such as radiolabelling, fluorescence-labelling, enzyme labelling and the like; preferably, enzyme labelling is employed, for instance by linking a peroxidase enzyme to the antibody.
  • the labelled antibody linked to the supported tubulin is detected by adding a substrate which is specific for the labelling enzyme.
  • the substrate is such that the enzyme transforms said substrate in a compound with different physicochemical characteristics, which are detectable by means of known analytical techniques.
  • suitable substrates are solutions containing compounds which develop a change in colour of the solution due to the enzymatic reaction, such as o-phenylenediamine or tetramethylbenzidine dihydrochloride; the intensity of the colour of the solution is determined by means of known photometric techniques, such as colorimetry, spectrophotometric and the like.
  • the first step of the present assay is the contact of the supported tubulin or tubulin-like peptide with a solution containing a proteolytic activity as above described.
  • a solution containing a proteolytic activity as above described.
  • the proteolytic solution contains in general from 0.005 to 50 ⁇ g/ml of the selected protease, preferably from 0.02 to 5 ⁇ g/ml, while the concentration of the inhibitor substance obviously varies according to the specific inhibition power of the substance, or may also be undetermined when an unknown inhibitor is investigated.
  • the protease may be pre-incubated together with the anti-proteolytic activity for a determined time at room temperature .
  • the incubation time of the proteolytic solution with the supported tubulin or tubulin-like peptide ranges from 1 to 5 hours, and is inversely proportional to the incubation temperature which is from 20°C to 40°C.
  • the anti-proteolytic activity is determined.
  • detection is carried out by using a labelled monoclonal antibody which specifically recognises the free end of the tubulin protein linked to the support.
  • the detection of the anti-proteolytic activity is performed by using: a) a monoclonal antibody which specifically recognises the C-terminus end of the tubulin protein; b) an enzyme labelled anti-antibody, specific for the above antibody; c) a substrate specific for the above labelling enzyme.
  • the detection of the anti-proteolytic activity is carried out by contacting the immobilized peptide with a solution containing a monoclonal antibody which specifically recognises the C-terminus of the ⁇ -tubulin protein, preferably a monoclonal rat antibody (e.g YL 1/2, Amersham); afterwards, a solution is added containing an enzyme labelled anti-antibody which is species specific for the first antibody, preferably a secondary peroxidated anti rat antibody when a monoclonal rat antibody has been employed in the previous step.
  • a monoclonal antibody which specifically recognises the C-terminus of the ⁇ -tubulin protein
  • a monoclonal rat antibody e.g YL 1/2, Amersham
  • the immobilized system tubulin - antibody - labelled antibody is then washed with a buffered solution and treated with a solution containing a specific substrate for the enzyme.
  • a substrate which develops a change in colour of the solution as a consequence of the enzymatic reaction is employed, such as o-phenylenediamine or tetramethylbenzidine dihydrochloride; as the intensity of the colour is proportional to the percentage of inhibition, it is possible to calculate a calibration curve by using known inhibitor solutions, thus determining the inhibition activity of unknown inhibitors. Furthermore it is possible to compare the different inhibition activity of a known inhibitor with respect to the different proteases.
  • the accuracy of the assay of the present invention is supported by the confirmation of the inhibition data of various known inhibitor substances.
  • pepstatin is a specific inhibitor for the proteases of the aspartic class, with different inhibition constants for the different aspartic proteases.
  • the 50% inhibition of cathepsin D and pepsin is obtained with a concentration of about 20 nM of pepstatin
  • 50% inhibition of renin is obtained with a concentration of about 200 nM of pepstatin
  • a concentration of about 2 ⁇ M is necessary to inhibit the Hiv-1 protease at 50%.
  • the test of the present invention allows also to confirm that the microbial alkaline inhibitor (MAPI) is able to inhibit a number of enzymes of the various protease classes, according to the data reported in literature (see for instance Watabe and Murao, Agric. Biol. Chem., 1979, 43-250).
  • MAMI microbial alkaline inhibitor
  • a further object of the present invention is an analytical kit for use in the method of the present invention.
  • Said kit comprises an analytical support bearing on its surface a covalently linked tubulin protein or a tubulin-like peptide and a solution containing a labelled monoclonal antibody which specifically recognises the free end of the tubulin protein linked to the support.
  • the preferred support to which the tubulin or tubulin-like peptide is linked the preferred way as to covalently link the tubulin or tubulin-like peptide to the support and the preferred labelled monoclonal antibody to be used for the detection of the inhibitor activity are those previously disclosed in the present application.
  • Microtubule protein is purified from calf brain through two cycles of temperature-dependent assembly and disassembly according to Islam K. and R.G Burns, FEBS Lett., 123, 1981, pp. 181-185.
  • Tubulin is separated from MAPs by applying the above purified protein on the top of a DEAE-Sephadex A-50 column (Pharmacia); the column is washed with PEM buffer solution containing 0.3M NaCl (to elute MAPs) prior to eluting with PEM buffer containing 0.50M NaCl and 0.1mM guanosine 5-tri-phosphate, according to the procedure described by Vallee et al. in "Methods in Enzymology", 134, 1986, pp. 89-116.
  • PEM buffer 1.1 1 of PIPES (piperazine-N,N'-bis(2-ethanesulphonic acid)-NaOH, pH 6.6, containing 1 mM of EGTA (ethylene glycol bis-( ⁇ -aminoethylether)-N,N,N',N'-tetraacetic acid) and 1 mg of MgSO 4 .
  • the title peptide is synthesized by conventional solid-phase methods using an ABI APPLIED BIOSYSTEMS@ Model 430-A Peptide Synthesizer and protocols supplied by the manufacturer.
  • the synthesis is carried out using a Boc-Tyr-(2-BrZ) resin (0.61 mmol/g, 0.5 mmol) and N-Boc amino acids with the following side chains protecting groups: Arg(Tos), His(Bom), Tyr(2-BrZ), Glu(Bzl).
  • Sequential coupling is performed using the standard Boc-peptide synthesis control, with DCC/HOBT in NMP. Boc-deprotection at each step is carried out with TFA.
  • the peptide is cleaved from the resin support and the side chains deprotected with anhydrous hydrogen fluoride/anisole (10:1) at 0°C. After removal of the HF the crude peptide is extracted from the resin with 30% acetic acid and lyophilized.
  • the peptidic material that remained is purified by reverse phase HPLC (Vydac C-18, 22x250 mm) using a Waters Deltaprep 3000 system, yielding 91 mg of pure peptide.
  • the peptide is characterized by analytical HPLC (Vydac 218TP54), FAB Mass Spectrometry and amino acid analysis:
  • PBS phosphate buffered saline
  • the covalent linkage of the peptide prepared according to example la to the support is obtained by following the procedure according to example 2, but using the peptide obtained according to example la instead of tubulin.
  • the employed proteolytic enzymes are:
  • the inhibitors tested are pepstatin (inhibitor of aspartic class), ⁇ -anti trypsin (inhibitor of the serine class), leupeptin (inhibitor of the cysteine class) and MAPI (non-specific inhibitor).
  • Table I reports the concentration of these inhibitors which determines a 50% inhibition of the above proteases.
  • a YL 1/2 antibody solution (Amersham) diluted 1:1000 in PBS buffer is added to each well and incubated for 1 hour at room temperature.
  • an anti-rat peroxydated antibody solution (Amersham) diluted 1:1000 in PBS buffer is added to each well and incubated for 1 hour at room temperature.
  • the reaction is stopped with H 2 SO 4 4 M.
  • the colour is detected on a Titertek Multi scan MCC340-HK spectrophotometer reading the absorbance at 492 nm. The results are reported in the following table I.
  • Concentration of inhibitor in the proteolytic solution which determines the 50% inhibition of the protease Proteases Conc. ( ⁇ g/ml) Concentration of inhibitor ( ⁇ g/ml) Pepstatin ⁇ -antitrypsin Leupeptin MAPI Trypsin 0.25 n.i. 1.0 n.i. 2.5 Subtilisin 0.25 n.i. 7.5 n.i. 4.0 Elastase 0.5 n.i. 1.0 n.i. n.i. Cathepsin G 5 n.i. n.i. n.i. n.i. Pepsin 5 0.03 n.i. n.i. 10 Cathepsin D 5 0.03 n.i. n.i.
  • Example 3 is repeated, but the proteolytic solution is pre-incubated with the inhibitor at room temperature for 5 minutes. The same results as in Example 3 are obtained.
  • Example 3 is repeated, but the solution containing the proteolytic activity and the known inhibitor is incubated for about 31 ⁇ 2 hours at 25°C on the supported tubulin. The same results as in Example 3 are obtained.

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Claims (14)

  1. Festphasenimmuntest zum Nachweis von proteolytischen Inhibitoren, umfassend:
    (a) das Inkontaktbringen eines Tubulin-Proteins oder eines Tubulin-ähnlichen Peptids, das kovalent an einen geeigneten Träger gebunden ist, mit einer Lösung, die eine proteolytische Aktivität zusammen mit einem Protease-Inhibitor enthält;
    (b) den Nachweis der Inhibitor-Aktivität gegenüber den ausgewählten Proteasen durch Inkontaktbringen des Trägers mit einer Lösung, die einen markierten monoclonalen Antikörper enthält, der spezifisch das freie Ende des an den Träger gebundenen Tubulin-Proteins erkennt.
  2. Immuntest nach Anspruch 1, worin das Tubulin-Protein oder das Tubulin-ähnliche Peptid über sein N-terminales Ende kovalent an den Träger gebunden ist.
  3. Immuntest nach Anspruch 1 oder 2, worin der Träger, an den das Tubulin-Protein oder das Tubulin-ähnliche Peptid gebunden ist, die Vertiefungen von aus Polymermaterialien hergestellten Mikrotiterplatten darstellt.
  4. Immuntest nach Anspruch 3, worin das Polymermaterial der Vertiefungen der Mikrotiterplatten Polyethylen, Polystyrol oder Polyvinylchlorid ist und das Tubulin an den Träger mit Hilfe eines Aktivierungsmittels gebunden wird, das ausgewählt ist aus Carbodiimid-Derivaten, N-Hydroxysuccinimid-Derivaten und Sulfosuccinimid-Derivaten.
  5. Immuntest nach Anspruch 4, worin das Aktivierungsmittel Bis(sulfosuccinimidyl)-suberat ist.
  6. Immuntest nach einem der Ansprüche 1 bis 5, worin der markierte monoclonale Antikörper ein System ist, das einen monoclonalen Antikörper und einen markierten anti-Antikörper umfaßt, worin der erste Antikörper spezifisch das freie Ende des gebundenen Tubulins erkennt, während der markierte anti-Antikörper spezifisch ist für den ersten Antikörper.
  7. Immuntest nach Anspruch 6, worin der anti-Antikörper mit einem Peroxidase-Enzym markiert ist.
  8. Immuntest nach Anspruch 6 oder 7, worin der monoclonale Antikörper spezifisch das C-terminale Ende von α-Tubulin erkennt.
  9. Immuntest nach einem der Ansprüche 1 bis 8, worin das an den Träger gebundene Tubulin-Protein α-Tubulin ist.
  10. Peptid, das die folgende Aminosäuresequenz, Seq ID No. 1, hat:
    Figure 00260001
  11. Verwendung des Peptids nach Anspruch 10 in dem Festphasenimmuntest nach einem der Ansprüche 1 bis 8.
  12. Analytischer Kit, enthaltend einen analytischen Träger, der an seiner Oberfläche ein Tubulin-Protein oder ein Tubulin-ähnliches Peptid aufweist, das über seinen N-Terminus kovalent gebunden ist, und eine Lösung, enthaltend einen markierten monoclonalen Antikörper, der spezifisch das C-terminale Ende des Tubulin-Proteins erkennt.
  13. Analytischer Kit nach Anspruch 12, worin das an den Träger gebundene Tubulin α-Tubulin ist.
  14. Analytischer Kit nach Anspruch 12, worin der markierte monoclonale Antikörper ein System ist, das einen monoclonalen Antikörper und einen markierten anti-Antikörper umfaßt, worin der erste Antikörper spezifisch den C-Terminus von α-Tubulin erkennt, während der markierte anti-Antikörper spezifisch ist für den ersten Antikörper.
EP95913069A 1994-03-29 1995-03-09 Festphasenimmunoassay zum nachweis von inhibitoren von einem proteolytischen enzym Expired - Lifetime EP0753152B1 (de)

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PCT/EP1995/000867 WO1995026505A1 (en) 1994-03-29 1995-03-09 Solid phase immunoassay to detect inhibitors of proteolytic enzymes

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ZA979542B (en) * 1996-11-14 1998-05-12 Rohm & Haas Use of certain amides as probes for detection of antitubulin activity and residence monitoring.
US20030068655A1 (en) * 2001-09-12 2003-04-10 Protiveris, Inc. Microcantilever apparatus and methods for detection of enzymes
US20100135855A1 (en) * 2008-11-26 2010-06-03 Koninklijke Philips Electronics N.V. Method for depositing substances on a support
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US5235039A (en) * 1991-06-10 1993-08-10 Eli Lilly And Company Substrates for hiv protease
US5288612A (en) * 1991-07-03 1994-02-22 The Scripps Research Institute Assay methods for detecting serum proteases, particularly activated protein C
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